A large number of diseases, such as cancer, birth defects, and infections can be identified and evaluated using nucleic acid screening. In some cases, the presence of a single mutation, e.g., BRCA1, is a strong indicator of a likelihood of developing disease. In other cases, a disease manifests with a combination of trace mutations, and the level of the mutant nucleic acids relative to the wild-type is indicative of the progression of the disease. In either case, techniques that allow detection of rare nucleic acid mutations with non-invasive sampling make it possible for subjects to be monitored regularly for the presence of the disease. Such monitoring allows for early intervention while avoiding unnecessary treatment. Ideally, such methods should be low-cost, to allow for regular monitoring of a large population of patients.
Standard nucleic acid separation techniques limit clinicians' abilities to analyze samples for nucleic acids that are present in low abundance, however. In particular, it is difficult to resolve rare nucleic acids that are present at low concentrations in the presence of closely-related nucleic acids, e.g., wild-type DNA. Furthermore, many non-invasive sampling methods, e.g., blood draws or buccal swabs, only provide a limited number of mutant nucleic acids, as compared to a tumor biopsy.
To resolve rare mutations in a sample, state-of-the-art methods typically amplify all of the nucleic acids prior to isolation and analysis. For example, using Polymerase Chain Reaction (PCR) amplification, each nucleic acid in a sample can be amplified one million times (or more). Theoretically, there will be a million-fold increase of each nucleic acid originally present, and, thus, a greater opportunity to isolate and find the nucleic acids in low abundance. In practice, however, PCR amplification has significant drawbacks when used to amplify nucleic acids that are present in low abundance. The PCR reaction is stochastic, and to the extent that a low-abundance nucleic acid is not amplified in the first few rounds of PCR, it likely will not be detected. In addition, PCR amplification introduces sequence errors in the amplicons. If the error rate is high enough, there can be a significant effect on the resulting sequence data, especially in applications requiring the detection of rare sequence variants. In fact, mutations present at a concentration on the order of the level of detection (LOD) of state-of-the-art techniques (about 1%) cannot be reliably determined because of the amplification errors introduced by PCR.
In addition to lacking the needed sensitivity, state-of-the-art nucleic acid screening techniques are also expensive, costing several thousand dollars to identify only a handful of biomarkers at a time. The high costs reflect that the techniques are technically challenging, time-consuming, and require the use of apparatus with limited availability. New methods of labeling nucleic acids, such as barcoding, allow multiplexed high throughput sequencing of samples, which can reduce the cost of an individual sample. Nonetheless, these labelling methods often rely on PCR amplification to incorporate the labels, and suffer many of the same problems, such as introduction of errant bases and unequal amplification due to early biases in regard to which nucleic acids are amplified.
Accordingly, there is still a need for techniques that easily isolate rare nucleic acids from a sample prior to further processing, e.g., sequencing. It would also be beneficial if such techniques could simultaneously process multiple nucleic acids, either from the same subject or from pooled subject samples.